Porin B (PorB) as a therapeutic target for prevention and treatment of infection by chlamydia

ABSTRACT

The present invention features the use of PorB polypeptide as a therapeutic agent. In specific embodiment the invention features a chlamydial vaccine based on a PorB polypeptide, as well as methods for induction of a protective immune response against infection by Chlamydia and Chlamydiophila. The invention further features methods for identifying agents that offset PorB function (e.g., in transport of α-ketoglutarate and which are effective as anti-chlamydial chemotherapeutic agents.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0001] This invention was made, at least in part, with a governmentgrant from the National Institutes of Health (Grant Nos. NIH grantsAI40250, AI39258, and AI42156). Thus, the U.S. government may havecertain rights in this invention.

FIELD OF THE INVENTION

[0002] The invention relates generally to the field of diagnosis,treatment, and prevention of infectious disease, particularly toprevention of infectious disease caused by the bacterial pathogenChlamydia and Chlamydophila (formerly classified as, for example, C.psittacci and C. pneumoniae).

BACKGROUND OF THE INVENTION

[0003] Chlamydiae are obligate intracellular pathogens that cause aspectrum of diseases including trachoma, the leading cause ofpreventable blindness worldwide, as well as a variety of sexuallytransmitted diseases such as lymphogranuloma venereum, urethritis,cervicitis, endometritis, and salpingitis (Thylefors et al. (1995) BullW H O 73:115-121). For example, Chlamydia trachomatis is considered theworld's most common sexually transmitted bacterial pathogen (Schachterand Grayston (1998) Presented at the Ninth international symposium onhuman chlamydial infection, Napa, Calif.; World Health Organization,1996, Global prevalence and incidence of selected curable sexuallytransmitted diseases: overview and estimates). Currently an estimated400 million people have active infectious trachoma, while 90 millionhave a sexually transmitted disease caused by C. trachomatis (WorldHealth Organization, 1996). Chlamydia pneumoniae usually infects thelungs and causes no more than a mild cold; however, it can travel to theblood vessels and thrive in clots, causing heart disease. Diseasescaused by Chlamydia represent significant health problems worldwide.

[0004] Growth of Chlamydia generally depends on the acquisition of hostATP and other high-energy metabolites from the host (Moulder et al.(1991) Microbiol. Rev. 55:143-90). Chlamydiae have the enzymaticmachinery for the Embden-Meyerhoff pathway (EMP), the pentose phosphatepathway (PPP), and the tricarboxylic acid (TCA) cycle (Kalman et al.(1999) Nat. Genet. 21:385-9; Stephens et al. (1998) Science 282:754-9).The TCA in chlamydia is incomplete in that the host lacks three enzymes:citrate synthase, aconitase, and isocitrate dehydrogenase (Kalman etal., ibid,; Stephens et al., ibid.). This observation suggests that theglutamate and α-ketoglutarate are obtained from the host cell sincethese can not be synthesized-by the bacterium. It has been shown thatchlamydiae utilize glucose as the major source of carbon, but thatdicarboxylates also serve to support chlamydial viability and growth((Iliffe-Lee et al. (2000) Mol. Microbiol. 38:20-30).

[0005] Treatment for Chlamydia infection typically involvesadministration of an antimicrobial drug such as azithromycin,doxycycline, ofloxacin, erythromycin, or amoxicillin (Centers forDisease Control and Prevention. Recommendations for the prevention andmanagement of Chlamydia trachomatis infections. Morb Mortal Wkly Rep1993; 42 (RR-12): 1-102). These conventional treatments are problematicfor several reasons, including patient non-compliance with multi-day,multi-dose regimens and side effects such as gastrointestinal problems.Furthermore, treatment of Chlamydia with existing antimicrobial drugsmay lead to development of drug resistant bacterial strains,particularly where the patient is concurrently infected with othercommon bacterial infections.

[0006] In addition, chlamydial infections often have no overt symptoms,so irreversible damage can be done before the patient is aware of theinfection. Therefore, prevention of the infection is considered the bestway to protect from the damage caused by Chlamydia. Therefore, thedevelopment and production of effective chlamydial vaccines, moreeffective treatments once infection is established, and sensitive andspecific diagnostic assays are important public health priorities.

[0007] Chlamydia have a unique developmental growth cycle withmorphologically distinct infectious and reproductive forms, elementarybodies (EB) and reticulate bodies (RB), respectively. The outer membraneproteins of EB are highly cross-linked with disulfide bonds. Thechlamydial outer membrane complex (COMC), which includes the major outermembrane protein (MOMP), is a major component of the chlamydial outermembrane. The COMC is made up of a number of cysteine-rich proteins(Everett et al. (1995) J. Bacteriol. 177:877-882; Newhall et al. (1986)Infect. Immun. 55:162-168; Sardinia et al. (1988) J. Gen. Microbiol.134:997-1004), as determined by the insolubility of proteins in the weakanionic detergent N-lauryl sarcosinate (Sarkosyl). Insolublity ofproteins in Sarkosyl is a characteristic of integral outer membraneproteins of gram-negative bacteria (Filip et al. (1973) J. Bacteriol.115:717-722). The COMC is present on the outer membrane proteins of EB,but not of RB. In contrast, MOMP is present throughout the developmentalcycle in both EB and RB and is thought to have a structural role due toits predominance and extensive disulfide crosslinking in the EBmembrane. Another function of MOMP is as a porin which allows fornon-specific diffusion of small molecules into Chlamydia (Bavoil et al.(1984) Infect. Immun. 44:479-485, Wyllie et al. (1998) Infect. Immun.66:5202-5207).

[0008] As with many pathogens, the development of a vaccine to Chlamydiahas proven difficult. Much of the focus for a vaccine candidate has beenon the chlamydial major outer membrane protein (MOMP) (see, e.g., U.S.Pat. Nos. 5,770,714 and 5,821,055; and PCT publication nos. WO 98/10789;WO 99/10005); WO 97/41889 (describing fusion proteins with MOMPpolypeptides); WO 98/02546 (describing DNA immunization based onMOMP-encoding sequences); WO 94/06827 (describing synthetic peptidevaccines based on MOMP sequences); WO 96/31236). MOMP has been estimatedto make up over 60% of the total outer membrane of Chlamydia and is anexposed surface antigen (Caldwell et al. (1981) Infect. Immun.31:1161-1176) with different sequence regions conferring serotype,serogroup and species reactivities (Stephens et al. (1988) J. Exp. Med.167:817-831). The protein consists of five conserved segments and fourvariable segments with the variable segments corresponding to surfaceexposed regions and conferring serologic specificity (Stephens et al.(1988) J. Exp. Med. 167:817-831). It has been suggested that thesevariable segments provide Chlamydia with antigenic variation, which inturn is important in evading the host immune response (Stephens, 1989Antigenic variation of Chlamydia trachomatis, p. 51-62. In J. W. Moulder(ed.), Intracellular Parasitism. CRC Press, Boca Raton.). A potentialproblem in making a vaccine to an antigenically variant region is that avaccine to one region of MOMP may only confer protection to thatserovar. Also, making a subunit vaccine to an antigenic variable regionmay prove difficult since conformational antigenic determinants may beessential to elicit effective immunization (Fan et al. (1997) J. Infect.Dis. 176:713-721). Although the use of MOMP as a vaccine still seemspromising, these potential problems strongly suggest that other vaccinetargets should be explored.

[0009] Other proposed Chlamydia vaccine targets have been described andinclude, for example, glycolipid exoantigen (see, e.g., U.S. Pat. Nos.5,840,297; 5,716,793 and 5,656,271). Other Chlamydia vaccines have usedother proteins (see, e.g, PCT publication no. WO 98/58953, describing asurface protein of C. pneumoniae) or a cocktail of proteins (see, e.g.,U.S. Pat. No. 5,725,863; and 5,242,686) or have used live or attenuatedwhole bacteria (see, e.g. U.S. Pat. Nos. 5,972,350; 4,267,170; and4,271,146). The sequencing of the genome of C. trachomatis has provideda tool to identify candidate vaccine targets (Stephens et al. (1998)Science 282:754-759) and examination of antibodies present in serum ofinfected individuals (Sanchez-Campillo et al. (1999) Electrophoresis20:2269-79) have provided tools for the identification of additionalvaccine targets.

[0010] There is a need in the field for the development ofchemotherapeutics and vaccines that provide protection against Chlamydiaand Chlamydiophila infection. The present invention addresses theseneeds.

SUMMARY OF THE INVENTION

[0011] The present invention features the use of PorB polypeptide as atherapeutic agent. In specific embodiment the invention features achlamydial vaccine based on a PorB polypeptide, as well as methods forinduction of a protective immune response against infection by Chlamydiaand Chlamydiophila. The invention further features methods foridentifying agents that offset PorB function (e.g., in transport ofα-ketoglutarate and which are effective as anti-chlamydialchemotherapeutic agents.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 is an alignment of the amino acid sequences of PorB (SEQ IDNO:2) and MOMP (SEQ ID NO:3).

[0013]FIG. 2 is a schematic showing the alignment of the amino acidsequences of PorB from C. trachomatis serovars D (CT-D) (SEQ ID NO:2),L2 (CT-L2) (SEQ ID NO:5), and C (CT-C) (SEQ ID NO:6), as well as theamino acid sequence of PorB from C. pneumoniae (CPn) (SEQ ID NO:4). C.trachomatis serovar L2 and C differences are indicated below the aminoacid sequence. The cysteines are indicated with an asterisk above theamino acid sequence.

[0014]FIG. 3 is a graph showing antibody neutralization of C.trachomatis serovar L2 in HaK cells. Results are expressed as percentagereduction in inclusion-containing cells with respect to number ofinclusion-containing cells observed after incubation with SPG only. Theantibodies used were anti-PorB (open circles), anti-PorB²⁴⁻⁷¹ (closedtriangles), IH5 (closed circles), anti-pgp3 (open squares) andpre-immune serum (closed squares).

[0015]FIGS. 4A and 4B are graphs showing the results of a liposomeswelling analysis of PorB (FIG. 4A) and the outer membrane of E. coliexpressing MOMP (panel B). Liposomes were made as described and 0.017 mlout of a total of 0.3 ml was diluted in 0.6 ml of isotonic sugarsolutions of stachyose (closed circles), sucrose (open squares), glucose(closed triangles) and arabinose (open circles). The y-axis represents arange of A₄₀₀ 0.15.

[0016] FIGS. 5A-5B are graphs showing lack of amino acid transportthrough PorB. Liposome swelling analysis of PorB (panel A) and the outermembrane of E. coli expressing MOMP (panel B). Liposomes were made asdescribed and 0.017 ml out of a total of 0.3 ml was diluted in 0.6 ml ofisotonic sugar solutions of stachyose, arabinose (open circles) andalanine (closed triangles). The y-axis represents a range of A₄₀₀ 0.15.

[0017]FIGS. 6A and 6B are graphs showing the results of a liposomeswelling analysis of PorB (panel A) and the outer membrane of E. coliexpressing MOMP (OmpA) (panel B). Isotonic solutions of stachyose(closed circles), arabinose (closed diamonds) and α-ketoglutarate (opentriangles). The y-axis represents a range of A₄₀₀ 0.15.

[0018]FIG. 7 is a graph showing the results of a liposome swelling assayto control for effects of ions that may be present in the test solute.Liposomes containing NAD⁺, stachyose, and imidazone-NAD were diluted inisotonic test solutions of citrate (closed circles), oxaloacetate(closed diamonds), and α-ketoglutarate (open triangles). The y-axisrepresents a range of A₄₀₀ 0.15.

[0019]FIG. 8 is a graph showing enzyme-linked liposome assay testing ofthe entry and oxidation of α-ketoglutarate. The formation of NADH usingliposomes containing PorB (closed circles) or lacking PorB (closedsquares) was measured by an increase in O.D.₃₄₀.

[0020]FIG. 9 is a graph showing liposome swelling analysis of PorB usingTCA-cycle intermediates. Isotonic sugar solutions of stachyose (closedcircles), arabinose (closed diamonds), α-ketoglutarate (open triangles),malate (closed squares) and succinate (open circles). The y-axisrepresents a range of A₄₀₀ 0.15.

[0021]FIG. 10 is a schematic showing the structures of compounds testedfor diffusion into liposomes containing PorB. Compounds in shaded boxeswere not efficiently transported by PorB.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0022] Before the present invention is described, it is to be understoodthat this invention is not limited to particular embodiments described,as such may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

[0023] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

[0024] It must be noted that as used herein and in the appended claims,the singular forms “a”, “and”, and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference to“a cell” includes a plurality of such cells and reference to “thepolynucleotide” includes reference to one or more polynucleotides andequivalents thereof known to those skilled in the art, and so forth.

[0025] The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

[0026] Definitions

[0027] As used herein, “immunoprotective response” is meant to encompasshumoral and/or cellular immune responses that are sufficient to: 1)inhibit or prevent infection by a microbial organism, particularly apathogenic microbial organism; and/or 2) prevent onset of disease,reduce the risk of onset of disease, or reduce the severity of diseasesymptoms caused by infection by a microbial organism, particularly apathogenic microbial organism.

[0028] As used herein the term “isolated” is meant to describe acompound of interest that is in an environment different from that inwhich the compound naturally occurs. “Isolated” is meant to includecompounds that are within samples that are substantially enriched forthe compound of interest and/or in which the compound of interest ispartially or substantially purified.

[0029] As used herein, the term “substantially purified” refers to acompound that is removed from its natural environment and is at least60% free, preferably 75% free, and most preferably 90% free from othercomponents with which it is naturally associated.

[0030] The term “artificial membrane” is meant to encompass a membranethat provides for incorporation of a functional PorB in the membrane(e.g. a PorB that can transport α-ketoglutarate), but which is not apart of a living organism (e.g., a liposome, a lipid bilayer that isformed in vitro and independent of a living cell, and the like).

[0031] By “subject” or “patient” or “individual” is meant any mammaliansubject for whom diagnosis or therapy is desired, particularly humans.Other subjects may include cattle, sheep (e.g., in detection of sheep atrisk of abortion due to chlamydial infection), dogs, cats (e.g., indetection of cats having eye and/or respiratory infections), birds (e.g.chickens or other poultry), guinea pigs, rabbits, rats, mice, horses,and so on. Of particular interest are subjects having or susceptible toChlamydia infection, particularly to infection by C. trachomatis, C.psittaci and/or C. pneumoniae.

[0032] The term “effective amount” or “therapeutically effective amount”means a dosage sufficient to provide for treatment for the disease statebeing treated or to otherwise provide the desired effect (e.g.,induction of an effective immune response or reduction of bacterialload). The precise dosage will vary according to a variety of factorssuch as subject-dependent variables (e.g., age, immune system health,etc.), the disease (e.g., the species of the infecting pathogen), andthe treatment being effected. In the case of an intracellular pathogeninfection, an “effective amount” is that amount necessary tosubstantially improve the likelihood of treating the infection, inparticular that amount which improves the likelihood of successfullypreventing infection or eliminating infection when it has occurred.

[0033] “Treatment” or “treating” as used herein means any therapeuticintervention in a subject, usually a mammalian subject, generally ahuman subject, including: (i) prevention, that is, causing the clinicalsymptoms not to develop, e.g, preventing infection and/or preventingprogression to a harmful state; (ii) inhibition, that is, arresting thedevelopment or further development of clinical, symptoms, e.g.,mitigating or completely inhibiting an active (ongoing) infection sothat bacterial load is decreased to the degree that it is no longerseriously harmful, which decrease can include complete elimination of aninfectious dose of a Chlamydia bacteria from the subject; and/or (iii)relief, that is, causing the regression of clinical symptoms, e.g.,causing a relief of fever, inflammation, and/or other symptoms caused byan infection.

[0034] As a point of clarification, it is noted that recently twospecies of bacterium, Chlamydia psittacci and Chlamydia pneumoniae, havebeen reclassified into the genus Chlamydophila. Unless specificallynoted otherwise, reference to the genus Chlamydia is meant to encompassall bacteria belonging to this genus, as well as the psittacci andpneumoniae species that have or soon may be reclassified asChlamydophila. Use of the terms “Chlamydia” and “chlamydial” are notmeant to be limiting to those bacterial species originally classifiedChlamydia, but are also meant to encompass the newly classified speciesof Chlamydophila as well unless specifically noted otherwise.

[0035] Overview

[0036] The invention is based on the expression and characterization ofa chlamydial outer membrane porin protein, PorB, of C. trachomatis, andthe further discovery that anti-PorB antibodies neutralizes theinfectivity of Chlamydia.

[0037] The inventors have discovered that PorB has severalcharacteristics that make it an effective vaccine and chemotherapeutictarget. Unlike other vaccine candidates such as MOMP, PorB does not varysubstantially in its amino acid sequence between serovars and wasinstead highly conserved among the C. trachomatis strains tested. Thislack of variable regions indicates that PorB does not participate inantigenic variation that contributes to invasion of the immune response.PorB sequences between C. trachomatis and C. pneumoniae are alsoconserved further supporting a requirement for constrained sequence toensure its specific function, and providing further evidence that avaccine based on a PorB polypeptide from one chlamydial species canprovide for immunoprotection across chlamydial species.

[0038] In addition, PorB facilitate transport of α-ketoglutarate intothe chlamydial bacterium. α-ketoglutarate feeds the chlamydialtricarboxylic acid (TCA) cycle, and is essential for providing thebacterium with carbon and energy production intermediates. Blocking ofPorB function, then can lead to arrest of bacterial cell growth and/orcell death. Thus, PorB is an attractive chemotherapeutic target.

[0039] The invention thus provides vaccines based upon PorB, and methodsof inducing anti-chlamydial immunity based on these vaccines. Inaddition, the invention also provides for detection of PorB polypeptidesor PorB-encoding sequences in diagnosis of Chlamydia infection.

[0040] The invention further features methods of identifyinganti-chlamydial chemotherapeutics based upon identification of agentsthat inhibit PorB function in α-ketoglutarate transport.

[0041] Specific aspects of the invention will now be described in moredetail.

[0042] Vaccines

[0043] In one aspect, the present invention provides a method ofinducing a protective immune response to infection by Chlamydia by useof a vaccine composition comprising an immunogenic PorB polypeptide.

[0044] The PorB polypeptide delivered to the host to elicit an immuneresponse may be a complete (e.g., native or full-length) PorBpolypeptide protein, or an immunoprotective portion (i.e., a portion ofthe PorB polypeptide sufficient to elicit a protective immune response)thereof. The PorB polypeptide can be the naturally-occurring form of theprotein, or an immunogenic, immunoprotective fragment thereof (i.e., afragment of PorB polypeptide that, upon administration to a host, canelicit an immune response, preferably an immunoprotective immuneresponse), a recombinant form of PorB polypeptide, a syntheticallyproduced PorB polypeptide or immunogenic fragment thereof, a modifiedrecombinant PorB polypeptide (e g, PorB polypeptide provided as a fusionprotein), a PorB polypeptide variant or analog that retainsimmunogenicity of native PorB or an immunogenic fragment thereof (e.g.an immunogenically similar or identical PorB-derived amino acidsequence), and the like. PorB polypeptide fragments of interest aregenerally from at least about 6 amino acids to about fragments of about8 amino acids, usually at least about 12 amino acids, more usually atleast about 20 amino acids, and generally at least about 50 to 100 aminoacids.

[0045] In one embodiment, the vaccine comprises a PorB polypeptide of C.trachomatis, C. pneumoniae or C. psittaci, preferably a PorB polypeptideof C. trachomatis. In one embodiment the C. trachomatis polypeptidecomprises an amino acid sequence of an immunogenic fragment of SEQ IDNO:2.

[0046] PorB polypeptide can be delivered to the host in a variety ofways including, but not limited to, delivery as an isolated orsubstantially purified protein preparation, by immunization with a PorBpolypeptide-encoding nucleic acid (e.g., by genetic immunizationtechniques known in the art), or by delivery of shuttle vector (e.g., aviral vector (e.g., a recombinant adenoviral vector), a recombinantbacterial vector (e.g., a live, attenuated heterologous bacterialstrain, e.g., live, attenuated Salmonella) that provides for delivery ofPorB polypeptide-encoding nucleic acid for expression in a host cell.Where nucleic acid encoding a PorB polypeptide is used in the vaccineformulation, the nucleic acid (e.g., DNA or RNA) can be operably linkedto a promoter for expression in a cell of the subject.

[0047] Formulation of Vaccine

[0048] The PorB polypeptide-based vaccine can be formulated in a varietyof ways. In general, the vaccine of the invention is formulatedaccording to methods well known in the art using suitable pharmaceuticalcarrier(s) and/or vehicle(s). A suitable vehicle is sterile saline.Other aqueous and non-aqueous isotonic sterile injection solutions andaqueous and non-aqueous sterile suspensions known to be pharmaceuticallyacceptable carriers and well known to those of skill in the art may beemployed for this purpose.

[0049] Optionally, a vaccinal composition of the invention may beformulated to contain other components, including, e.g., adjuvants,stabilizers, pH adjusters, preservatives and the like. Such componentsare well known to those of skill in the vaccine art.

[0050] Administration of Vaccine

[0051] The PorB polypeptide vaccine is administered in an “effectiveamount,” that is, an amount of PorB polypeptide or PorBpolypeptide-encoding nucleic acid that is effective in a route ofadministration to elicit an immune response effective to facilitateprotection of the host against infection by Chlamydia. For example,where PorB polypeptide is delivered using a nucleic acid construct or arecombinant virus, the nucleic acid construct or recombinant virus isadministered in an amount effective for expression of sufficient levelsof the selected gene product to provide a vaccinal benefit, i.e.,protective immunity.

[0052] Conventional and pharmaceutically acceptable routes ofadministration include intranasal, intramuscular, intratracheal,subcutaneous, transdermal, subdermal, intradermal, topical, rectal, oraland other parental routes of administration. Routes of administrationmay be combined, if desired, or adjusted depending upon the immunogen orthe disease. The vaccine composition can be administered in a singledose or in multiple doses, and may encompass administration of boosterdoses, to elicit and/or maintain immunity. Methods and devices foraccomplishing delivery are well known in the art. For example foradministration through the skin, any of a variety of transdermal patchescan be used to accomplish delivery.

[0053] The amount of PorB polypeptide, PorB polypeptide-encoding nucleicacid, or PorB polypeptide recombinant virions in each vaccine dose isselected as an amount which induces an immunoprotective response withoutsignificant, adverse side effects in typical vaccines. Such amount willvary depending upon which specific immunogen is employed, whether or notthe vaccine is adjuvanted, and a variety of host-dependent factors.Where PorB polypeptide protein is delivered directly, it is expectedthat each does will comprise 1-1000 μg of protein, generally from about1-200 μg, normally from about 10-100 μg. An effective dose of a PorBnucleic acid-based vaccine will generally involve administration of fromabout 1-1000 μg of nucleic acid. An optimal amount for a particularvaccine can be ascertained by standard studies involving observation ofantibody titres and other responses in subjects. The levels of immunityprovided by the vaccine can be monitored to determine the need, if any,for boosters. Following an assessment of antibody titers in the serum,optional booster immunizations may be desired. The immune response tothe protein of this invention is enhanced by the use of adjuvant and oran immunostimulant.

[0054] Subjects

[0055] Using the methods and compositions described herein in connectionwith the subject invention, an immunoprotective response againstchlamydial infection can be induced in any subject, human or non-human,susceptible to infection by a chlamydial strain, particularly achlamydial strain pathogenic for the subject species. In general, themethods of the invention are effective in preventing or inhibitinginfection of a Chlamydia species that expresses on its surface a proteinthat is immunocrossreactive with PorB. In one embodiment administrationof a PorB polypeptide of C. trachomatis induces an immune responseprotective against infection by C. trachomatis, C. pneumoniae and C.psittaci, particularly an immunoprotective response against C.trachomatis. In another embodiment administration of a PorB polypeptideof C. pneumoniae induces a protective immune response against infectionby C. trachomatis, C. pneumoniae and C. psittaci, and particularly animmunoprotective response against C. pneumoniae. In another embodiment,administration of a PorB polypeptide of C. psittaci induces a protectiveimmune response against infection by C. trachomatis, C. pneumoniae andC. psittaci and particularly an immunoprotective response against C.psittaci.

[0056] Human disease associated with chlamydial infection that can bemitigated or prevented using the methods and compositions describedherein include, but are not necessarily limited to, sexually transmitteddisease (urethritis and epidiymitis in men; pelvic inflammatory diseasein women), conjunctivitis, and pneumonia Of particular interest is theinhibition or prevention of infection by C. trachomatis, by C.pneumonia, and by C. psittaci. Exemplary chlamydial diseases aredescribed in more detail below.

[0057]C. trachomatis, the most common cause of sexually transmitteddiseases in the United States, causes a variety of diseases includingnongonococcal urethritis and epididymitis in men; cervicitis,urethritis, and pelvic inflammatory disease in women; Reiter's syndrome;and neonatal conjunctivitis and pneumonia, the latter of which aregenerally acquired through maternal transmission. C. trachomatis hasbeen implicated in 20% of adults with pharyngitis. Several immunotypesof C. trachomatis can cause lymphogranuloma venereum (LGV), a diseasefound mostly in tropical and subtropical areas. LGV strains invade andreproduce in regional lymph nodes.

[0058]C. pneumoniae (previously called Taiwan acute respiratory agent orTWAR), originally considered a serotype of C. psittaci, can causepneumonia, especially in children and young adults. The organism hasbeen found in atheromatous lesions, and infection is associated withincreased risk of coronary artery disease.

[0059]C. psittaci infects many animals, but human infection is closelyrelated to contact with birds. In humans, C. psittaci causespsittacosis, an infectious a typical pneumonia transmitted to humans bycertain birds. In humans, psittacosis (ornithosis, parrot fever) isusually caused by inhaling dust from feathers or excreta of infectedbirds or by being bitten by an infected bird; rarely, it occurs byinhaling cough droplets of infected patients or venereally.Human-to-human transmission may be associated with highly virulent avianstrains.

[0060] Where the subject is non-human, subjects of particular interestinclude feline, bovine, and avian subjects.

[0061] Anti-PorB Antibodies

[0062] Antibodies that specifically bind a PorB polypeptide can beadministered to provide temporary, passive immunity against chlamydialinfections. Methods for production of anti-PorB antibodies (e.g.,monoclonal-or polyclonal antibodies) are well known in the art, as aremethods for formulating such antibodies for administration. i oneembodiment the anti-PorB antibody is a humanized antibody. Anti-PorBantibodies of interest also encompass modified antibodies (e.g.,modified to increase biological half-life following administration).

[0063] Anti-PorB antibodies are administered in an amount sufficient toneutralize Chlamydia so as to prevent, mitigate, or reduce thelikelihood of onset of infection. Anti-PorB antibodies can beadministered by any suitable route, generally by parenteral injection(e.g. subcutaneous, intramuscular, intravenous, etc.). Administration ofanti-PorB antibodies particularly useful for preventing or inhibitinginfection in immunocompromised subjects or other subjects having animmune system that can not maintain an effective, immunoprotectiveresponse to, for example, a PorB antigen.

[0064] Antibodies that bind PorB of two or more Chliamydial species in amanner effective to block infection by each of these species are ofparticular interest. For example, antibodies that bind PorB of C.trachomatis as well as PorB of C. pneumoniae and/or C. psittaci, can beused to provide passive immunity that protects against infection by eachof these chlamydial species.

[0065] Diagnosis of Chlamydial Infection

[0066] In addition to the uses in vaccines described above, PorBpolypeptides and sequences obtained from PorB-encoding polynucleotidescan be used in the detection of Chlamydia infection in a subject and/ordetermining whether a subject has been exposed to Chlamydia infection.Diagnostic assays based upon detection of PorB or a PorB-encodingsequence in a biological sample include, but are not necessarily limitedto, detection of PorB polypeptides, detection of anti-PorB antibodies,and/or detection of PorB-encoding sequences in a test sample from thesubject. Detection of any of these PorB polypeptide, polynucleotides, orantibodies in a sample taken from a subject is indicative of chlamydialinfection in the subject. For clarity, applicants note that “probe” asused herein in the context of detection of PorB polypeptides or PorBpolypeptide-encoding polynucleotides is meant to encompass anti-PorBantibodies (e.g. for detection of PorB polypeptide), PorB polypeptide orfragments thereof (e.g., for detection of anti-PorB antibodies), andPorB polynucleotides and fragments thereof (e.g., for use inhybridization or PCR assays to detect PorB polynucleotides).

[0067] In one general embodiment, the methods of the invention involvesdetecting PorB polypeptides or anti-PorB antibodies in the subject bycontacting an appropriate biological test sample from a subjectsuspected having been exposed to Chlamydia or suspected of having aChlamydia infection with a probe that is one of a) an antibody thatspecifically binds a PorB polypeptide, b) a PorB polypeptide. After thetest sample is contacted with the probe for a time sufficient to allowfor formation of specific antibody-PorB polypeptide binding pairs, theformation of such binding pairs is detected. Detection of theantibody-PorB binding pairs indicates that the subject has been exposedto Chlamydia (due to the presence of PorB polypeptides in the samplewhere the probe is an anti-PorB antibody), or has mounted an immuneresponse to PorB polypeptide (due to the presence of anti-PorBantibodies in the sample, as detected using a PorB polypeptide probe)which suggests at least prior exposure to Chlamydia.

[0068] In another general embodiment, the presence of a Chlamydiainfection in the host is detected using an probe to specificallyhybridizes to and/or specifically amplifies (e.g. through use of PCR) toa PorB polypeptide encoding polynucleotide. Detection of hybridizationor detection of a specific PCR product indicates that the subject has aChlamydia infection.

[0069] Diagnosis Based on Detection of PorB Polypeptides and/oranti-PorB Antibodies

[0070] Detection of PorB polypeptides can be accomplished according to awide variety of immunoassays that are well known in the art, and may beperformed either qualitatively or quantitatively. For example, thediagnostic assay can measure the reactivity between an anti-PorBantibody (e.g., a polyclonal or monoclonal antibody (MAb), preferably aMAb) and a patient sample, usually a sample of a bodily fluid, e.g.,mucosal secretion, blood-derived sample (e.g., plasma or serum), urine,and the like. The patient sample may be used directly, or diluted asappropriate, usually about 1:10 and usually not more than about1:10,000. Immunoassays may be performed in any physiological buffer,e.g. PBS, normal saline, HBSS, PBS, etc.

[0071] In one embodiment, a conventional sandwich type assay is used. Asandwich assay is performed by first immobilizing proteins from the testsample on an insoluble surface or support. The test sample may be boundto the surface by any convenient means, depending upon the nature of thesurface, either directly or indirectly. The particular manner of bindingis not crucial so long as it is compatible with the reagents and overallmethods of the invention. They may be bound to the plates covalently ornon-covalently, preferably non-covalently.

[0072] The insoluble supports may be any compositions to which the testsample polypeptides can be bound, which is readily separated fromsoluble material, and which is otherwise compatible with the overallmethod of detecting and/or measuring type I cell- or type IIcell-specific polypeptide. The surface of such supports may be solid orporous and of any convenient shape. Examples of suitable insolublesupports to which the receptor is bound include beads, e.g. magneticbeads, membranes and microtiter plates. These are typically made ofglass, plastic (e.g. polystyrene), polysaccharides, nylon ornitrocellulose. Microtiter plates are especially convenient because alarge number of assays can be carried out simultaneously, using smallamounts of reagents and samples.

[0073] Before adding patient samples or fractions thereof, thenon-specific binding sites on the insoluble support, i.e. those notoccupied by polypeptide, are generally blocked. Preferred blockingagents include non-interfering proteins such as bovine serum albumin,casein, gelatin, and the like. Alternatively, several detergents atnon-interfering concentrations, such as Tween, NP40, TX100, and the likemay be used.

[0074] Samples, fractions or aliquots thereof can be added to separatelyassayable supports (for example, separate wells of a microtiter plate).A series of standards, containing known concentrations of PorB can beassayed in parallel with the samples or aliquots thereof to serve ascontrols and to provide a means for quantitating the amounts of PorBpolypeptide present in the test sample. Preferably, each sample andstandard will be added to multiple wells so that mean values can beobtained for each.

[0075] After the test sample polypeptides are immobilized on the solidsupport, anti-PorB antibody is added. The incubation time of the sampleand the antibody should be for at time sufficient for antibody bindingto the insoluble polypeptide. Generally, from about 0.1 to 3 hr issufficient, usually 1 hr sufficing.

[0076] After incubation, the insoluble support is generally washed ofnon-bound components. Generally, a dilute non-ionic detergent medium atan appropriate pH, generally 7-8, is used as a wash medium. From one tosix washes may be employed, with sufficient volume to thoroughly washnon-specifically bound proteins present in the sample. After washing,antibody binding to the sample can be detected by virtue of a detectablelabel on the antibody. Where the antibody is not detectably labeled,antibody binding can be detected by contacting the sample with asolution containing antibody-specific second receptor, in most cases asecondary antibody (i.e., an anti-antibody). The second receptor may beany compound which binds antibodies with sufficient specificity suchthat the bound antibody is distinguished from other components presentIn a preferred embodiment, second receptors are antibodies specific forthe anti-PorB antibody, and may be either monoclonal or polyclonal sera,e.g. goat anti-mouse antibody, rabbit anti-mouse antibody, etc.

[0077] The antibody-specific second receptors are preferably labeled tofacilitate direct, or indirect quantification of binding. Examples oflabels which permit direct measurement of second receptor bindinginclude light-detectable labels, radiolabels (such as ³H or ¹²⁵I);fluorescers, dyes, beads, chemiluminescers, colloidal particles, and thelike. Examples of labels which permit indirect measurement of bindinginclude enzymes where the substrate may provide for a colored orfluorescent product. In a preferred embodiment, the second receptors areantibodies labeled with a covalently bound erzyme capable of providing adetectable product signal after addition of suitable substrate. Examplesof suitable enzymes for use in conjugates include horseradishperoxidase, alkaline phosphatase, malate dehydrogenase and the like.Where not commercially available, such antibody-enzyme conjugates arereadily produced by techniques known to those skilled in the art.

[0078] Alternatively, the second receptor may be unlabeled. In thiscase, a labeled second receptor-specific compound is employed whichbinds to the bound second receptor. Such a second receptor-specificcompound can be labeled in any of the above manners. It is possible toselect such compounds such that multiple compounds bind each molecule ofbound second receptor. Examples of second receptor/secondreceptor-specific molecule pairs include antibody/anti-antibody andavidin (or streptavidin)/biotin. Since the resultant signal is thusamplified, this technique may be advantageous where only a small amountof PorB polypeptide is present, or where the background measurement(e.g., non-specific binding) is unacceptably high. An example is the useof a labeled antibody specific to the second receptor. Morespecifically, where the second receptor is a rabbit anti-allotypicantibody, an antibody directed against the constant region of rabbitantibodies-provides a suitable second receptor specific molecule. Theanti-Ig will usually come from any source other than human, such asovine, rodentia, particularly mouse, or bovine.

[0079] The volume, composition and concentration of anti-antibodysolution provides for measurable binding to the antibody already boundto receptor. The concentration will generally be sufficient to saturateall antibody potentially bound to PorB polypeptide. The solutioncontaining the second receptor is generally buffered in the range ofabout pH 6.5-9.5. The solution may also contain an innocuous protein aspreviously described. The incubation time should be sufficient for thelabeled ligand to bind available molecules. Generally, from about 0.1 to3 hr is sufficient, usually 1 hr sufficing.

[0080] After the second receptor or second receptor-conjugate has bound,the insoluble support is generally again washed free of non-specificallybound second receptor, essentially as described for prior washes. Afternon-specifically bound material has been cleared, the signal produced bythe bound conjugate is detected by conventional means. Where an enzymeconjugate is used, an appropriate enzyme substrate is provided so adetectable product is formed. More specifically, where a peroxidase isthe selected enzyme conjugate, a preferred substrate combination is H₂O₂and is O-phenylenediamine which yields a colored product underappropriate reaction conditions. Appropriate substrates for other enzymeconjugates such as those disclosed above are known to those skilled inthe art. Suitable reaction conditions as well as means for detecting thevarious usefull conjugates or their products are also known to thoseskilled in the art. For the product of the substrate O-phenylenediaminefor example, light absorbance at 490-495 nm is conveniently measuredwith a spectrophotometer.

[0081] The absence or presence of antibody binding may be determined byvarious methods that are compatible with the detectable label used,e.g., microscopy, radiography, scintillation counting, etc. Generallythe amount of bound anti-PorB antibody detected will be compared tocontrol samples (e.g., positive controls containing PorB or negativecontrols lacking such polypeptides). The presence of anti-PorB antibodyis indicative of the presence of a Chlamydia in the test sample, whichin turn is indicative of chlamydial infection in the subject.

[0082] As will be readily appreciated by the ordinarily skilled artisanupon reading the present disclosure, the above techniques can be readilymodified to provide for detection of anti-PorB antibodies in the host.For example, rather than immobilizing PorB polypeptide on a solidsupport, an anti-PorB antibody is immobilized on the support andsubsequently contacted with a test sample from the host. Binding of PorBpolypeptide from the test sample to the support-bound anti-PorB antibodycan then be detected using a second anti-PorB antibody (e.g., that bindsto a different epitope of the polypeptide than the bound antibody).Binding of the second antibody can then be detected according to methodswell known in the art.

[0083] Diagnosis Based on Detection of PorB Nucleic Acid

[0084] Where the diagnostic assay involves detection of a PorB-encodingsequence, the assay can take advantage of any of a variety ofpolynucleotide detection techniques that are well known in the art. Forexample, a fragment of a PorB-encoding sequence can be used as a probeto detect hybridizing sequences in a test sample, or for use as a primerin PCR amplification of chlamydial nucleic acid in at test sample.Methods for detecting sequences based on hybridization, as well as useof PCR are known in the art, see, e.g., Sambrook, et al. MolecularCloning: A Laboratory Manual, CSH Press 1989. The probe or primer maycomprise a detectable label. Suitable labels include fluorochromes, e.g.fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerytirin,allophycocyanin, 6-carboxyfluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX),6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), radioactivelabels, e.g. ³²P, ³⁵S, ³H; etc. The label may be a two stage system,where the polynucleotide is conjugated to biotin, haptens, etc. having ahigh affinity binding partner, e.g. avidin, specific antibodies, etc.,where the binding partner is conjugated to a detectable label. In PCR,the label may be conjugated to one or both of the primers;alternatively, the pool of nucleotides used in the amplification islabeled, so as to incorporate the label into the amplification product.

[0085] Kits for Detection of Chlamydia

[0086] PorB polypeptides (for detection of specific antibodies),anti-PorB antibodies, and PorB polynucleotide probes and/or primers, aswell as other materials useful in the diagnostic methods of theinvention (e.g., labels, compounds for detection of labels, solidsupports for capture of nucleic acid in a sample, filters for at leastpartial separation or purification of parasites in the sample,detergents and other reagents (e.g., lysing mammalian and cells in thesample), etc.) can be provided in a kit. Such kits can include samplesto serve as positive controls or negative controls. Preferably such kitsare designed for use in the field, e.g., do not contain components thatrequire refrigeration, are portable, etc.

[0087] PorB as a Chemotherapeutic Target and Identification ofAnti-Chlamydial Agents

[0088] Also of interest are candidate agents that affect PorB expression(e.g. by affecting PorB promoter function) or that interact with PorBpolypeptides. Agents of interest can include those that inhibit PorBactivity. PorB activity can be decreased by, for example, decreasing theefficiency of α-ketoglutarate transport by PorB, associating with theporin to inhibit α-ketoglutarate transport, decreasing transcription ortranslation of the PorB gene product, and the like. Agents that decreasePorB activity can be used to, for example, treat Chlamydia infection ina subject. The agent can be selected for chemotherapeutic activityagainst chlamydia either extracellularly, intracellulary, or both.

[0089] “Candidate agents” is meant to include synthetic molecules (e.g.,small molecule drugs, peptides, or other synthetically producedmolecules or compounds, as well as recombinantly produced gene products)as well as naturally-occurring compounds (e.g., polypeptides, factorsendogenous to a prokaryotic or eukaryotic host cell, plant extracts, andthe like). Of particular interest are candidate agents that can crossthe cell membrane of the host cell for the treatment of intracellularbacteria.

[0090] Candidate agents encompass numerous chemical classes, thoughtypically they are organic molecules, preferably small organic compoundshaving a molecular weight of more than 50 and less than about 2,500daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including, but not limited to: peptides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof.

[0091] Candidate agents are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fingal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

[0092] Drug Screening Assays

[0093] In general, drug screening identifies agents that reverse orinhibit PorB function and may provide a means to treat a Chlamydiainfection in a subject. Of particular interest are screening assays foragents that have a low toxicity for subject cells and are able to crossthe cell membrane of subject cells. Generally a plurality of assaymixtures are run in parallel with different agent concentrations toobtain a differential response to the various concentrations. Typically,one of these concentrations serves as a negative control, i.e. at zeroconcentration or below the level of detection.

[0094] Screening of Candidate Agents in vitro

[0095] A wide variety of in vitro assays may be used to screen agentsfor their ability to affect PorB activity, including screening forantibacterial (e.g., bactericidal, bacteriostatic, etc.) activity in achlamydial culture (e.g., in an infected cell line, etc.), labeled invitro binding assays (e.g., competitive binding assays, and the like),immunoassays for protein binding, liposome-swelling assays (e.g.,liposomes having incorporated PorB), and the like. For example, byproviding for the production of large amounts of PorB protein, one canidentify ligands or substrates that bind to the protein. The purifiedprotein may also be used for determination of three-dimensional crystalstructure, which can be used for modeling intermolecular interactions(e.g. to model interaction of the porin with α-ketoglutarate to providethe basis of rational drug design).

[0096] The screening assay can be a binding assay, wherein one or moreof the molecules may be joined to a label, and the label directly orindirectly provides a detectable signal. Various labels includeradioisotopes, fluorescers, chemiluminescers, enzymes, specific bindingmolecules, particles, e.g. magnetic particles, and the like. Specificbinding molecules include pairs, such as biotin and streptavidin,digoxin and antidigoxin etc. For the specific binding members, thecomplementary member would normally be labeled with a molecule thatprovides for detection, in accordance with known procedures.

[0097] A variety of other reagents may be included in the screeningassays described herein. Where the assay is a binding assay, theseinclude reagents like salts, neutral proteins, e.g. albumin, detergents,etc that are used to facilitate optimal protein-protein binding,protein-DNA binding, and/or reduce non-specific or backgroundinteractions. Reagents that improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.may be used.

[0098] The mixture of components are added in any order that providesfor the requisite binding.

[0099] Incubations are performed at any suitable temperature, typicallybetween 4 and 40° C. Incubation periods are selected for optimumactivity, but may also be optimized to facilitate rapid high-throughputscreening. Typically between 0.1 and 1 hours will be sufficient.

[0100] PorB-encoding nucleic acid can be introduced and expressed in aprokaryotic recombinant host cell (e.g., E. coli, and the like) so toprovide for production of functional PorB in the bacterial outermembrane. The PorB-expressing recombinant host cell can then becontacted with candidate agents and control substrates known to diffusethrough PorB, e.g. α-ketoglutarate, succinate, oxaloacetate, arabinose,glucose, glutamate, adipate, malonate, etc., preferably α-ketoglutarate,and the effect of the candidate agent on PorB function in transport ofthe control substances tested.

[0101] For example, the candidate agent or the control substance can bedetectably labeled, and the effect of the candidate agent monitored bymeans of the detectable label. For example, wherein the controlsubstrate is detectably labeled, the ability of the candidate agent toinhibit PorB transport of the control substrate can be monitored byexamining depletion of the detectable label from the extracellular mediaor by examining the level of intracellular detectable label, or both.

[0102] In one embodiment, the ability of an agent to modulate PorBfunction is evaluated using PorB within an artificial membrane. In oneexample, the artificial membrane is provided in the context of aliposome. In a specific embodiment, the assay is based on a liposomeswelling assay (see, e.g., Nikaido (1983) Methods Enzymol. 97:85-95;Nikaido and Rosenberg (1983) J. Bacteriol. 153:241-252). Briefly, PorBis cloned and expressed in a suitable cell line, e.g. E. coli.,purified, and incorporated into liposomes. The liposomes can then becontacted with candidate agents and control substrates known to diffusethrough PorB, e.g. α-ketoglutarate, succinate, oxaloacetate, arabinose,glucose, glutamate, adipate, malonate, etc., and liposome swellingmeasured, for example, by following the change in O.D.₄₀₀ using aPerkin-Elmer spectrophotometer and an attached chart recorder. Alteredliposome swelling as compared to a control liposome contacted with onlythe substrate indicates that the candidate agent can modulate PorBfunction. Of particular interest are agents that inhibit PorB functionas evidenced by reduced liposome swelling as compared to a control.

[0103] The screening assays of the invention can be supplemented by, ormodified to, identify agents that can enter into an infected eukaryoticcell, where it can exhibit its anti-chlamydial effect upon intracellularbacteria. This can be accomplished by, for example, contacting candidateagents, particularly those pre-screened for their inhibition of PorBfunction, with mammalian cells infected with chlamydia, and assessingthe effect of the agent upon growth of the intracellular bacteria (e.g.,by assessing affect upon growth rate, bacterial load in the host cell,and the like).

[0104] Other variations on the screening assay to identify agents thataffect PorB function are within the scope of the present invention. Forexample, a polynucleotide encoding PorB or a modified PorB polypeptide(e.g., PorB fusion polypeptide or other modified PorB polypeptide thatis adapted for expression in and insertion into the extracellularmembrane of a eukaryotic host cell) can be introduced into a eukaryotic(e.g., mammalian) host cell (e.g. in an isolated cell in vitro, is e.g.,in a mammalian cell line) for expression using methods well known in theart. Recombinant mammalian cells producing PorB can then be screened forfunction in transport of the natural ligand (e.g., α-ketoglutarate) andused as the basis of an assay to identify agents that inhibitα-ketoglutarate transport.

[0105] Identified Candidate Agents

[0106] The compounds having the desired pharmacological activity may beadministered in a physiologically acceptable carrier to a host fortreatment of a disease associated with Chlamydia infection, e.g.sexually transmitted diseases, conjunctivitis, pneumonia, etc. Thetherapeutic agents may be administered in a variety of ways, orally,topically, parenterally e.g. subcutaneously, intraperitoneally,intravascularly, etc. Inhaled treatments are also of interest. Dependingupon the manner of introduction, the compounds may be formulated in avariety of ways. The concentration of therapeutically active compound inthe formulation may vary from about 0.1-100 wt. %.

[0107] The pharmaceutical compositions can be prepared in various forms,such as granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing Agents, wetting and emulsifying Agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.

EXAMPLES

[0108] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the present invention, and are not intended to limitthe scope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

[0109] Methods and Materials

[0110] The following procedures are used in the Examples described indetail below. Although some of the methods described below are in commonuse, the specific protocol used in the Examples below is described indetail where alternative protocols are often employed. Basic proceduressuch as DNA digestion by restriction enzymes and ligation are notdescribed, as such are well within the skill of the ordinarily skilledartisan and, in some instances, are carried out according to the enzymeor kit manufacturer's instructions.

[0111] Chlamydial cultures. C. trachomatis strains B/TW-5/OT, C/TW-3/OT,and L2/434/Bu were grown in L929 cells, and strain D/UW-3/Cx was grownin HeLa 229 cells. Elementary bodies (EB) and reticulate bodies (RB)were separately purified by diatrizoate (Renograffin; E. R. Squibb andSons, Princeton, N.J.) gradients and were used immediately afterpurification or stored at −70 C. RB was purified at 24 hourspost-infection.

[0112] Bacterial strains and plasmid. The synthetic gene encoding MOMP(ompA) was constructed in E. coli HMS 174 (DE3) and has been previouslydescribed (Jones et al. (2000) Gene 258:173-181). E. coli HMS 174 (DE3)without the plasmid was used as a control strain. PorB cloning andexpression were done in the E. coli strain, TOP10 (Invitrogen, Carlsbad,Calif.). The complete PorB gene was cloned into the pBAD-TOPO TA vector(nvitrogen), which contains the araBAD promoter.

[0113] Expression and purification of PorB. Regulation of expression isby the AraC gene product on the promoter in the absence or presence ofarabinose. All E. coli cultures were grown with aeration at 37° C. inLuria-Bertani broth containing 100 mg/ml of ampicillin until thecultures reached an O.D of 0.6. 0.02% Arabinose to a final concentrationof 0.5 mM was added to induce the expression of PorB. PorB was clonedwith a C-terminal HIS tag and purified by nickel column using the HISBind Purification system (Novagen, Madison, Wis.). Extraction of PorBwith 1% octylglucoside at 37° C. for 1 h and dialysis of the detergentout of the extracted PorB using PBS and then I X Bind buffer (Novagen)was necessary before purification by nickel column. IPTG was added to afinal concentration of 0.5 mM to induce the expression of MOMP. Theouter membranes of E. coli expressing MOMP were purified as described inMOMP Jones et al. (2000 Gene 258:173-181)

[0114] Outer membrane preparation. The spheroplasts and outer membranesof E. coli were isolated using the method of Osborn and Munson (1974Methods Enzymol. 0.31:642-653) with the following modifications. The E.coli were grown in Luria Bertani broth with 100 mg/ml ampicillin at 37°C. with vigorous aeration to a density of approximately 5×10⁸bacteria/ml, followed by 2 hours of induction by addition of 0.02%arabinose to a concentration of 0.5 mM. 25 ml aliquots of thespheroplasts were lysed by sonication by immersing in an ice-salt bathand sonicating for three 15-second periods with a Braunsonic Usonicator. The suspension was cooled for 1 minute between bursts. Theunbroken cells were removed by centrifugation at 1200× g for 15 minutesat 4° C. The supernatant fraction was then centrifuged for 2 hours at100,000× g at 4° C. The membrane pellet was resuspended in a smallvolume of cold 0.25 M sucrose-3.3 mM Tris-1 mM EDTA, pH 7.8 andcentrifuged for 2 hours at 100,000× g 4° C. The pellet was thensuspended in 6 ml of cold 25% sucrose-5 mM EDTA, pH 7.5 for separationby isopycnic centrifugation. An outer membrane preparation was performedwith a control clone expressing a non-outer membrane protein and thisprotein was not detected in the outer membrane fraction.

[0115] Chlamydial outer membrane complex (COMC) preparation. The COMCwas prepared from fresh, not previously frozen, purified EB (10 mg) andperformed according to the method of Caldwell et al. (Caldwell et al.(1981) Infect. Immun. 31:1161-1176) with some modifications. EB weresuspended in 3 ml of 10 mM sodium phosphate buffer (pH 7.4) and 2%Sarkosyl. This suspension was sonicated briefly and centrfuged at100,000× g for 1 hour at 20° C. Both the soluble and insoluble (COMC)fractions were analyzed by SDS-PAGE.

[0116] Antibodies. Polyvalent monospecific antisera to PorB wereobtained from mice Swiss-Webster mice immunized with 1) nickelcolumn-purified PorB protein and 2) a piece of PorB consisting of theamino-terminal portion, from amino acid 24-71 (PorB²⁴⁻⁷¹). The mice wereimmunized twice at two-week intervals with 100 μg of purified protein inan equal volume of complete Freund's adjuvant for the secondimmunization. IH5 is a L2 serovar specific monoclonal antibody specificto MOMP. Polyvalent antiserum produced in rabbits using L2 EB andpolyvalent monospecific antiserum produced in rabbits using cloned andexpressed 28 kDa plasmid protein (pgp3) (Comanducci et al. (1993) J.Gen. Microhiol. 139:1083-1092) were used in the dot blot experiment.

[0117] Cell staining. C. trachomatis serovar L2-infected, D-infected anduninfected HeLa cells were fixed in methanol for 10 minutes and washedthree times in PBS. The anti-PorB monospecific antibody was diluted1:200, added to the cells and incubated for 1 hour at room temperatureon a rocker platform. The monolayer was rinsed three times in PBS andoverlaid with a fluorescein isothiocyanate-conjugated anti-mouseimmunoglobulin G (Zymed, So. San Francisco, Calif.) diluted 1:50. Thecells were incubated in the dark for one hour at room temperature on arocker platform and then washed three times with PBS. The cells werethen counter stained with Evans blue and observed by fluorescencemicroscopy.

[0118] Dot Blot assay. Dot blots were performed as previously describedby Zhang et al., (1987 J. Immunol. 138:575-581) with the followingdifferences: 1) the method of detection was enhanced chemilurninescence(ECL) (Amersham Pharmacia Biotech, Piscataway, N.J.); 2) an anti-mouseHRP-conjugated secondary antibody was used; 3) the primary and secondaryantibodies were washed by rinsing the wells with PBS and discarding thePBS. Vacuum filtration was used after the final wash to remove allliquid from the wells.

[0119] Dot blots of viable chlamydial EB to determine surfaceaccessibility of PorB were performed by probing immobilized EB with (1)a negative control antibody, anti-pgp3; (2) a positive controlmonoclonal, IH5; (3) an anti-PorB antibody; (4) an anti-PorB²⁴⁻⁷¹antibody; and (5) a positive control polyclonal, anti-L2 EB was used.The anti-pgp3 antibody was used at 1:1000 for the immunoblot and bound a28 kDa protein, while it was used at 1:100 for the dot blot. The rabbitanti-L2 EB polyclonal antibody was used at 1:1000 for both theimmunoblot and dot blot. The IH5 monoclonal antibody was used at 1:1000for the immunoblot and at 1:4000 for the dot blot. The anti-PorBantibody was used at 1:200 for the immunoblot and at 1:100 for the dotblot.

[0120] Protease cleavage. Fresh EB, not previously frozen, wereincubated with various concentrations of trypsin (0, 0.001, 0.01, 0.1mg/ml) and proteinase K (0, 0.1, 0.5, 1 mg/ml) for 30 minutes at 37° C.The treated EB were then immediately transferred to a nitrocellulosemembrane and a dot blot analysis was performed as described above.

[0121] Neutralization assay. The HaK (hamster kidney cells) in vitroneutralization assay was performed as previously described (Byrne et al.(1993) J. Infect. Dis. 168:415-20). Antibodies, except for pre-immuneserum, were quantitated and diluted to 200 mg/ml, then serially dilutedby two-fold to 12.5 mg/ml. Pre-immune serum was used at a dilution of1:10 and serially diluted 2-fold to 1:160. For detection of PorB,monospecific anti-PorB was purified with protein A (Sigma, St. Louis,Mo.), filter sterilized, quantitated using the BCA assay (Pierce,Rockford, Ill.), and diluted in SPG to the appropriate concentrations. Acontrol monoclonal antibody with specificity for MOMP (IH5) was used.Also, a control monoclonal antibody with unrelated specificity, theanti-pgp3 antibody as well as the pre-immune serum were used ascontrols. L2 EB was diluted in SPG to contain 2×10⁴ IFU/ml, 100 ml wasadded to each antibody dilution in total volume of 200 ml.Neutralization proceeded for 30 minutes. IFU were quantitated bycounting ten fields at a magnification of 40×. A mean IFU per field wascalculated and the results were shown as percent reduction in mean IFUcompared with the control plates.

[0122] Quantitation of protein. Purified protein and outer membranes foruse in the liposome swelling assay was quantitated according to theLowry method. All other samples were quantitated by the BCA assay(Pierce, Rockford, Ill.).

[0123] Liposome swelling assay. The liposome swelling assay wasperformed according to the method of Nikaido (Nikaido & Rosenberg (1983)J. Bacteriol. 153:241-252) with the following modifications: 1)liposomes were made by mixing 5.0 pmol phosphatidylcholine and 0.02 μmoldicetylphosphate with outer membrane proteins or purified protein inorder to increase the optical density readings to the range of 0.4-0.7O.D., and 2) the liposome drying time was longer than 2 minutes (i.e., 5minutes), but at a lower temperature of 37° C. Liposomes were made witheither dextran T-40 (15% dextran T40 in 5 mM Tris-Cl, pH 7.5) orstachyose inside. Since stachyose is impermeable to the porins, it wasused as a control to determine the isoosmotic concentration of othersolutes. The concentration of stachyose which produced no swelling orshrinking of the proteoliposomes was determined to be the isoosmoticconcentration. The swelling rates were determined as d(1/OD)/dt from theoptical density changes between 10 and 20 seconds (Nikaido & Rosenberg(1983) J. Bacteriol. 153:241-252).

[0124] Liposome swelling assay for testing anions. Liposomes were madeaccording to the method described above with a few modifications. Thefollowing was added to phosphatidylcholine and dicetylphosphate driedwith PorB (6 μg): 4 mM NAD⁺, 12 mM stachyose, 1 mM imidazole-NAD bufferpH (6.0). The test solution consisted of 1 mM Imidazole-NAD (pH 6.0), 1mM Sodium NAD, 6 mM disodium salt of the anion to be tested(α-ketoglutarate, succinate, oxaloacetate, malate, or citrate). Controlliposomes without protein were used to determine the isotonicconcentration of the test solutions.

[0125] Enzyme-Iinked liposome swelling assay. Liposomes were made asdescribed above with addition of 50 mM potassium phosphate, 2.5 mM NAD⁺,0.2 mM thiamin pyrophosphate, 1.0 mM magnesium chloride, 0.13 mMcoenzyme A, 2.6 mM cysteine, and 5.0 units of α-ketoglutaratedehydrogenase. Various concentrations of α-ketoglutarate (0.001 mM-1 mM)were used as test solutes. Liposomes containing PorB (6 μg) and controlliposome without protein were made with the reaction mixture, washedthrough a Sephadex column (S-300) equilibrated with reaction nixturewithout α-ketoglutarate dehydrogenase, and placed inside a cuvette.α-ketoglutarate was added to the reaction and mixed. The formation ofNADH was measured by the increase in O.D.₃₄₀.

Example 1 Analysis of PorB Sequence—Comparison to Major Outer MembraneProtein (MOMP)

[0126] Genome sequence analysis revealed a number of predicted outermembrane proteins (see Stephens et al. 1998 “Genome sequence of anobligate intracellular pathogen of humans: Chlamydia trachomatis”Science 282:754-759). One such predicted outer membrane protein, encodedby the predicted open reading frame CT713, was selected for analysis,and referred to herein as PorB. The nucleotide and amino acid sequencesof PorB (CT713) are available within the complete sequence of the genomeat GenBank Accession No. NC_(—)000117, with the amino acid sequence atGenBank Accession No. gi|3329169. The open reading frame correspondingto PorB is the complement of nucleotide residues 3616 to 4638 of GenBankAccession No. AE001342. The nucleotide and amino acid sequences of PorBof C. trachomatis are provided in the Sequence Listing as SEQ ID NOS:1and 2, respectively. Alignment of the amino acid sequence of PorB withthe amino acid sequence of MOMP is provided in FIG. 1.

[0127] As illustrated in FIG. 1, PorB has only slight sequencesimilarity (20.4%) to MOMP. Despite this relatively low amino acidsequence similarity, PorB and MOMP do share certain characteristics andstructural features. The estimated size of this protein is 38,000daltons and the isoelectric point was calculated to be 4.9. MOMP has amolecular weight of 40,000 with an isoelectric point calculated andexperimentally confirmed to be 5.0 (Bavoil et al. (1984) Infect. Immun.44:479485). PorB has a predicted cleavable leader sequence as well as anamino acid sequence which ends in phenylalanine (arrow in FIG. 2), acharacteristic of many outer membrane protein (Struyve et al. (1991) J.Mol. Biol. 218:141-148). Both PorB and MOMP have the same number ofcysteines (9 cysteins) suggesting that PorB may be an outer membranecysteine-rich protein analogous to, although distinct from, MOMP.

[0128] Previous reports on outer membrane proteins of Chlamydia have notidentified this protein. The overabundance of MOMP and similarity insize and isoelectric point likely contributed in preventing earlierdetection of PorB. PorB is not as predominant as MOMP by approximately20-fold. Since PorB is similar in size to MOMP, an SDS-PAGE analysis ofchlamydial outer membrane complexes can not distinguish PorB from MOMP.Also, PorB has a similar isoelectric point to MOMP, therefore a 2-D gelanalysis may not separate the proteins (Bavoil et al. (1984) Infect.Immun. 44:479-485; Bini et al. (1996) Electrophoresis 17:185-190).

Example 2 Analysis of PorB Sequence—Comparison of PorB Amino AcidSequences from Different Serovars

[0129] When compared with other serovars of C. trachomatis, MOMP hasfour distinct variable segments which correspond to surface exposedregions of the protein. Serovar designations have been related to thedifferences in these variable segments of MOMP (Stephens et al. (1988)J. Ep. Med. 167:817-831). In order to determine whether this serovarvariation is also characteristic for PorB, the sequence ofPorB betweenserovars was compared.

[0130]FIG. 2 provides an alignment of the amino acid sequences of PorBfrom the C. trachomatis serovars D (CT-D) (SEQ ID NO:2), L2 (CT-L2) (SEQID NO:5), and C (CT-C) (SEQ ID NO:6), as well as the amino acid sequenceof PorB from C. pneumoniae (CPn) (SEQ ID NO:4). The PorB of C.trachomatis and C. pneumoniae are 59.4% identical. C. trachomatisserovar L2 and C differences are indicated below the amino acidsequence. The cysteines are indicated with an asterisk above the aminoacid sequence.

[0131] The nucleotide and amino acid sequence alignments betweenserovars D, B, C and L2 revealed no to only minor differences. There isno PorB sequence difference between serovars D and B, while there is onenucleotide difference, which results in an amino acid change, betweenserovars D (or B) and C. Between serovars D (or B) and L2 there are sixnucleotide differences, each of which result in a difference in theencoded amino acid. The nucleotide differences occur throughout the geneand were not clustered to any region (FIG. 2). Among the serovarsinvestigated, there are no variable segments in PorB such as there arein MOMP. Thus, sequence variation is not a phenotype for PorB.

[0132] Comparison between PorB of C. trachomatis (serovar D) and C.pneumoniae reveals greater differences dispersed throughout the gene.However, with 59.4% identity between anino acid sequences of C.trachomatis and C. pneumoniae, this protein is highly conserved betweenspecies (FIG. 2). C. pneumoniae PorB has 6 cysteines, four of which areconserved between species, while C. trachomatis serovars D, B and C have9 conserved cysteines and serovar L2 has 8.

Example 3 Expression of PorB in E. coli

[0133] PorB was predicted to be in the outer membrane through a varietyof protein localization programs such as PSORT (K. Nakai, Human GenomeCenter, Institute for Medical Science, University of Tokyo, Japan). Aleader sequence cleavage site for C. trachomatis PorB was predicted tobe at amino acid 26 (FIG. 2). The complete gene including the leadersequence was cloned into E. coli with a HIS tag at the C-terminal end ofPorB- and expressed. The protein was affinity purified by nickel columnchromatography. PorB expressed in E. coli was localized to the outermembrane fraction as determined by an immunoblot using an antibody tothe C-terminal HIS tag. E. coli porins were also detected in this outermembrane fraction by Coomassie stain. The presence of PorB was primarilylocalized to the outer membrane suggesting that PorB has the necessarysignal(s) to be transported to the outer membrane by E. coli.

Example 4 Presence of PorB in Inclusions

[0134] In order to characterize PorB in Chlamydia, a polyclonalmonospecific serum was produced to the complete purified protein. FITCcell staining experiments using the anti-PorB serum showed that thisserum contained antibody that bound antigens localized to the inclusionsin infected cells. Anti-PorB serum did not label uninfected controlcells. Staining cells infected with serovar L2 and serovar D, 48 and 72hours post infection, respectively with anti-PorB serum revealedpunctate staining consistent with the morphology for EB and RB. Thisantibody staining was present at 10, 15, 20, 24, 48 hours postinfection, indicating that this protein is constitutively expressedand/or present throughout the chlamydial development cycle.

Example 5 Localization of PorB to the COMC

[0135] The anti-PorB antibody bound a protein in Chlamydia that wassimilar in size to MOMP by immunoblot analysis. The amount of PorBpresent in EB and RB was similar. The serum also bound the purifiedHIS-tagged protein, which was detected by an anti-HIS antibody. Althoughthere were only slight similarities in sequence to MOMP, testing forcross reactivity between antibodies to PorB and MOMP was perfoimedAnti-PorB serum did not bind MOMP expressed in E. coli. Therefore, it isconcluded that the anti-PorB sera bound PorB and did not cross reactwith MOMP.

[0136] In order to determine if PorB is a component of the ChlamydiaOuter Membrane Complex (COMC), the COMC was isolated and probed withanti-PorB serum. Since the chlamydial outer membrane is highly disulfidebonded, the Sarkosyl insoluble fraction contains a number of proteinssuch as MOMP and other cysteine rich proteins. PorB was detected in theCOMC fraction and not the soluble supernatant. Therefore, the presenceof PorB in the COMC fraction demonstrates that this protein is in thechlamydial outer membrane and is disulfide linked perhaps to other COMCproteins.

Example 6 Surface Accessibility of PorB

[0137] Since PorB was predicted to be in the outer membrane. and waslocalized to the COMC, surface accessibility of this protein was tested.Dot blot experiments have been shown to be specific for surfaceaccessible antigens (Zhang et al. (1987) J. Immunol. 138:575-581) andwas used to test surface accessibility of PorB. The dot blot using theanti-PorB sera showed that this antibody bound EB. A negative controlrabbit polyclonal serum to a 28 kDa plasmid protein (pgp3) was used as anegative control antibody since this protein is not present in the outermembrane of Chlamydia (Comanducci et al. (1993) J. Gen. Microbiol.139:1083-1092). This negative control antibody did not bind EB, while apositive control antibody to a surface accessible antigen on MOMP (IH5)bound. These data demonstrate that PorB is localized to the outermembrane.

Example 7 Effect of Proteolytic Cleavage on PorB

[0138] To investigate surface exposure of PorB, purified EB weredigested with proteases and proteins from EB were assessed for bindingby the anti-PorB antibody. Using the dot blot method, EB were treatedwith various concentrations of trypsin or proteinase K, immobilized on anitrocellulose membrane and probed with the anti-PorB antibody, as wellas to antibodies to MOMP and the anti-pgp3 antibody. A reduction inbinding by anti-PorB antibodies was observed for EB-digested proteinssuggesting that PorB has surface accessible trypsin and proteinase Kcleavage sites, and thus is an outer membrane protein.

Example 8 Neutralization of C. trachomatis by Anti-PorB

[0139] Since PorB is an outer membrane protein with surface exposedregions, antibodies made to PorB were tested for ability neutralizeinfectivity of C. trachomatis (serovar L2). The anti-PorB sera producedusing either the entire protein or an amino-terminal fragment (aminoacids 24-71) at a concentration of 100 mg/mil neutralized infectivity byup to 88% and 70%, respectively, further supporting the conclusion thatPorB is a surface exposed outer membrane protein (FIG. 3). The controlantibody without specificity to outer membrane proteins, anti-pgp3, aswell as the pre-immune sera did not neutralize infectivity (FIG. 3). Amonoclonal antibody to serovar L2 MOMP (IH5) at a concentration of 50and 100 mg/ml neutralized infectivity up to 78% (FIG. 3). Thisneutralization assay confirms that antibodies to PorB can inhibitinfectivity by C. trachomatis since this assay is an art-recognized invitro correlate for the assessment of protective immunity (Byrne et al.(1993) J. Infect. Dis. 168:415-20).

Example 9 Pore-Forming Activity of PorB

[0140] The pore-forming capabilities of PorB were tested using theliposome reconstitution assay (Nikaido (1983) Methods Enzymol.97:85-95). The liposome swelling assay for study of porin function isused not only because it is well established, but because this assaygives precise information on the rates of diffusion of solutes throughthe porin channels (Nikaido & Rosenberg (1983) J. Bacteriol.153:241-252). This assay involves the formation of liposomesincorporated with pore-forming protein and then determination of whetherand how fast test solutes can diffuse through the protein channels. Thisassay was used to test and compare pore-forming activity of the C.trachomatis PorB and MOMP.

[0141] Purification of MOMP using mild detergents causes a loss in porinactivity (Bavoil, et al. (1984) Infect. Immun. 44:479-485, Wyllie, etal. (1998) Infect. Immun. 66:5202-5207), therefore, MOMP was expressedin E. coli and outer membrane fractions enriched for MOMP were used. Ithas been shown in liposome swelling assays that the predominant porinactivity of the outer membrane fraction of E. coli expressing MOMP isdue to MOMP (Jones et al. (2000) Gene 258:173-181). This was also foundto be the case for PorB except purified PorB also functioned in liposomeswelling assays (FIG. 4) and was used in all subsequent experiments. Tocontrol for potential contaminants that may occur during PorBpurification, another predicted outer membrane protein from C.trachomatis serovar D (CT241) was cloned, expressed in E. coli andpurified by the same procedure used for PorB. Like PorB, CT241 alsocontains a predicted leader sequence and ends in phenylalanine and wasincorporated into liposomes and tested for pore forming activity. Thisprotein as well as liposomes without protein did not show pore-formingactivity with any of the solutes tested.

[0142] The smallest sugars tested in the liposome swelling assay werethe monosaccharides arabinose and glucose. These sugars penetrated thePorB- and MOMP-containing liposomes faster than the disaccharide,sucrose, while the tetrasaccharide, stachyose, was too large to enter(FIG. 4). This diffusion selectivity of PorB- or MOMP-containingliposomes with sugars was similar to what has been observed withCOMC-containing liposomes (Bavoil, et al. (1984) Infect. Immun.44:479-485, Wyllie, et al. (1998) Infect. Immun. 66:5202-5207). Largersolutes enter into PorB or MOMP porin slower, suggesting that there is asize restriction of molecules that can enter via these porins. However,the liposomes containing PorB permitted the diffusion of arabinose orglucose at a slower rate than liposomes containing MOMP.

[0143] Since Chlamydia have been proposed to utilize amino acids fromhost cells (Ossowski et al (1965) Isr. J. Med. Sci. 1:186-193; Hatch etal. (1982) J. Bacteriol. 150:379-385; Pearce, (1986) Ann. Inst. PasteurMicrobiol. 137A:325-332), diffusion of amino acids through PorB and MOMPwere tested using the liposome swelling assay. MOMP liposomes allow forthe diffusion of all of the amino acids at different rates basedpredominantly on size selectivity and alanine and glycine enter throughMOMP liposomes slightly faster than arabinose (Jones et al. (2000) Gene258:173-181). In contrast, PorB liposomes did not efficiently allow forany of 20 amino acids to enter liposomes including the small amino acidssuch as alanine (FIG. 5). These data indicate that PorB is lessefficient than MOMP as a non-specific porin.

Example 10 Permeability of Solutes Through PorB

[0144] PorB was purified by nickel column chromatography andincorporated into liposomes. Liposomes enriched for MOMP were used tocompare the pore-forming activity of PorB. As shown above, PorB porinfunction, unlike MOMP, is inefficient in the diffusion of amino acids,even amino acids smaller in molecular weight than arabinose, such asglycine and alanine. MOMP porin activity is detected using only 1 μg ofprotein (total outer membrane protein) while 6-10 μg of purified PorB isneeded to observe comparable porin activity. This suggests that PorB ismuch less efficient as a non-specific porin or that the purificationprocess may have resulted in a less functional protein.

[0145] Differences in general pore-forming activity, as well asdifferences in the amount present in the chlamydial outer membrane,suggest a unique role for each of the porins. The presence of PorB insmall amounts is difficult to understand unless PorB has a role as asubstrate-specific porin that is efficient in the uptake of particularclasses of molecules. RT-PCR analysis and cell staining at various timepoints indicated that this protein is expressed throughout thedevelopmental cycle. Thus PorB expression is not differentiallyregulated.

[0146] In order to determine if PorB had specificity for anymolecule(s), the genome sequence was studied to determine if theinferred biology of Chlamydia could provide an idea of which moleculesChIamydia might need to obtain from the host. This analysis provided alist of orthologs of transporters that are important in thetranslocation of solutes across the inner membrane, including aminoacid, polysaccharide, oligopeptide, and dicarboxylate transporters(Stephens et al. (1998) Science 282:754-759). Previous analysis of MOMPporin activity showed that amino acids, mono- and di-saccharide andoligopeptides enter efficiently through MOMP (Jones et al. (2000) Gene258:173-181). However, PorB did not allow for the efficient entry ofeither amino acids or polysaccharides. The presence of an ortholog to aninner membrane dicarboxylate transporter, and that Chlamydia appears tohave a truncated TCA cycle, suggest that chlamydiae may requireexogenous α-ketoglutarate from the host cell. Therefore, the hypothesisthat dicarboxylates could enter through the chlamydial outer membranewas tested by measuring α-ketoglutarate diffusion through the two knownporins, PorB and MOMP.

[0147] The liposome swelling assay with PorB and MOMP showed that thediffusion of α-ketoglutarate was more efficient through PorB than MOMP(FIG. 6). No diffusion of α-ketoglutarate was seen with liposomeswithout protein, as well as liposomes with another chlamydial outermembrane protein (Omp85) that was purified by the same method as-PorB.Chlamydial Omp85 was used as a control protein that was cloned,expressed in E. coli and purified by the same method used to purifyPorB. E. coli not expressing PorB, which was treated the same way as E.coli expressing PorB, was purified by nickel column chromatography andthe column eluate was used as a control in all of the assays to verifythat no E. coli contaminants were responsible for the porin activityobserved.

[0148] One concern with the liposome assays was the possible influenceof ions present in anionic solutes, such as α-ketoglutarate, that maycause ion fluxes potentially confounding the results of the assay. Aliposome assay to control for the possibility of ion fluxes (Nikaido andRosenberg (1983) J. Bacteriol. 153:241-252) was used to confirm theswelling assay results. Liposomes were made with NAD-imidazole andstachyose to counteract any ion fluxes that may result from the presenceof contaminating ions in the α-ketoglutarate solute used for the assay.This assay confirmed that the results in the initial liposome assayswere not the result of ion fluxes and that oxaloacetate also enteredefficiently through PorB while citrate did not enter (FIG. 7).

[0149] An enzyme-linked liposome assay was used to further show that theα-ketoglutarate was entering through PorB. The liposomes were made withα-ketoglutarate dehydrogenase and NAD⁺ inside and washed The substrate,α-ketoglutarate, was added to the outside of the liposomes and then theliposomes were measured for the formation of NADH by the increase in theO.D.₃₄₀. This shows that α-ketoglutarate entered through PorB unlike thecontrol liposomes which did not allow α-ketoglutarate to enter insideand result in the formation of NADH (FIG. 8).

Example 11 TCA Cycle Molecules Enter Through PorB

[0150] Since α-ketoglutarate efficiently entered through PorB, a numberof other TCA cycle intermediates were tested to assess whether thisporin was specific for the α-ketoglutarate substrate. Succinate (andoxaloacetate) enter PorB with similar rates to α-ketoglutarate; however,malate did not enter efficiently (FIG. 9). Citrate did not enter throughPorB.

Example 12 Permeability Specificity Studies with PorB

[0151] Since dicarboxylates of the TCA cycle were tested and diffusedthrough PorB, other molecular analogues were studied to determine thecapability of PorB to distinguish between related molecules (FIG. 10). Adifference in carbon-chain lengths represented by adipate, glutarate,succinate, and malonate did not show marked differences in diffusioncompared to α-ketoglutarate, although 6-carbon adipate and 3-carbonmalonate entered through PorB at a slightly slower rate. Thus PorB didnot discriminate between different substrate chain lengths. The effectsof small side groups using analogues that differed only by specific sidegroups were tested. For example, α-ketoglutarate and glutarate enteredthrough PorB efficiently, but not glutamate that is similar instructure. The presence of the amino group seems to retard the diffusionof glutamate and this likely explains why other amino acids do not enterinto PorB efficiently. A comparison of 5-carbon compounds citrate andaconitate with only the addition of a hydroxyl group to citrateprevented the entry of citrate through PorB. Four-carbon malate andsuccinate also differ by the presence of a hydroxyl group and thediffusion rate was retarded for malate. Therefore, PorB can discernbetween very similar compounds to allow for specific selectivity,suggesting a substrate-specific selective porin. These findings showthat PorB facilitates the diffusion α-ketoglutarate and other selectdicarboxylates to enter chlamydial outer membranes efficiently.

[0152] While the present invention has been described with reference tothe specific embodiments thereof, it should be understood by thoseskilled in the art that various changes may be made and equivalents maybe substituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1 6 1 1023 DNA C. trachomatis CDS (1)...(1020) 1 atg agt agc aag cta gtgaac tat ctc cgt ttg act ttc cta tct ttt 48 Met Ser Ser Lys Leu Val AsnTyr Leu Arg Leu Thr Phe Leu Ser Phe 1 5 10 15 tta ggg atc gca tct acttca tta gac gct atg cct gcg ggg aat ccg 96 Leu Gly Ile Ala Ser Thr SerLeu Asp Ala Met Pro Ala Gly Asn Pro 20 25 30 gcg ttt cca gtc atc ccg gggatt aat att gaa cag aaa aat gcc tgt 144 Ala Phe Pro Val Ile Pro Gly IleAsn Ile Glu Gln Lys Asn Ala Cys 35 40 45 tct ttc gat tta tgt aat tct tatgat gta cta tcc gca ctg tcc ggt 192 Ser Phe Asp Leu Cys Asn Ser Tyr AspVal Leu Ser Ala Leu Ser Gly 50 55 60 aac ctg aag ctc tgc ttc tgc gga gattat atc ttt tca gaa gaa gct 240 Asn Leu Lys Leu Cys Phe Cys Gly Asp TyrIle Phe Ser Glu Glu Ala 65 70 75 80 cag gta aaa gat gtc cct gtc gtt acctct gtg aca aca gct ggg gtt 288 Gln Val Lys Asp Val Pro Val Val Thr SerVal Thr Thr Ala Gly Val 85 90 95 ggt cct tct cct gat att act tcg aca accaaa acg cga aat ttc gat 336 Gly Pro Ser Pro Asp Ile Thr Ser Thr Thr LysThr Arg Asn Phe Asp 100 105 110 ctc gtg aac tgt aat ctc aat aca aac tgtgta gct gta gct ttt tcc 384 Leu Val Asn Cys Asn Leu Asn Thr Asn Cys ValAla Val Ala Phe Ser 115 120 125 ctt cct gat cgt tcg ctg agc gcg att cctctg ttt gat gtg agt ttc 432 Leu Pro Asp Arg Ser Leu Ser Ala Ile Pro LeuPhe Asp Val Ser Phe 130 135 140 gaa gtg aaa gta gga gga ctg aaa caa tactac cgc ctt ccc atg aat 480 Glu Val Lys Val Gly Gly Leu Lys Gln Tyr TyrArg Leu Pro Met Asn 145 150 155 160 gcc tat cga gac ttc acc tcg gaa cctctc aat tct gaa tca gaa gtt 528 Ala Tyr Arg Asp Phe Thr Ser Glu Pro LeuAsn Ser Glu Ser Glu Val 165 170 175 acg gac ggg atg att gaa gta cag tccaat tac gga ttt gtt tgg gat 576 Thr Asp Gly Met Ile Glu Val Gln Ser AsnTyr Gly Phe Val Trp Asp 180 185 190 gtt agc ttg aaa aaa gtc ata tgg aaagat ggc gtt tcc ttt gta ggc 624 Val Ser Leu Lys Lys Val Ile Trp Lys AspGly Val Ser Phe Val Gly 195 200 205 gtc ggt gca gac tat cgc cat gct tcttgc cct att gac tac atc att 672 Val Gly Ala Asp Tyr Arg His Ala Ser CysPro Ile Asp Tyr Ile Ile 210 215 220 gca aac agt caa gct aat cca gaa gtattc atc gct gac tcg gat ggg 720 Ala Asn Ser Gln Ala Asn Pro Glu Val PheIle Ala Asp Ser Asp Gly 225 230 235 240 aaa ctg aac ttc aag gag tgg agtgtc tgc gta ggt ctt act acc tat 768 Lys Leu Asn Phe Lys Glu Trp Ser ValCys Val Gly Leu Thr Thr Tyr 245 250 255 gtg aat gac tac gtt ctt cct tactta gcg ttt tct ata ggg agt gtt 816 Val Asn Asp Tyr Val Leu Pro Tyr LeuAla Phe Ser Ile Gly Ser Val 260 265 270 tct cgc caa gct ccg gac gac agcttc aaa aaa tta gaa gat cgc ttc 864 Ser Arg Gln Ala Pro Asp Asp Ser PheLys Lys Leu Glu Asp Arg Phe 275 280 285 act aac ctc aaa ttt aaa gtt cgtaaa att acc agc tct cat cgt gga 912 Thr Asn Leu Lys Phe Lys Val Arg LysIle Thr Ser Ser His Arg Gly 290 295 300 aac atc tgc atc gga gcg aca aactat gtc gcc gat aac ttc ttc tac 960 Asn Ile Cys Ile Gly Ala Thr Asn TyrVal Ala Asp Asn Phe Phe Tyr 305 310 315 320 aac gta gaa gga aga tgg ggaagc cag cgc gct gtg aac gtc tcc gga 1008 Asn Val Glu Gly Arg Trp Gly SerGln Arg Ala Val Asn Val Ser Gly 325 330 335 gga ttc caa ttc taa 1023 GlyPhe Gln Phe 340 2 340 PRT C. trachomatis 2 Met Ser Ser Lys Leu Val AsnTyr Leu Arg Leu Thr Phe Leu Ser Phe 1 5 10 15 Leu Gly Ile Ala Ser ThrSer Leu Asp Ala Met Pro Ala Gly Asn Pro 20 25 30 Ala Phe Pro Val Ile ProGly Ile Asn Ile Glu Gln Lys Asn Ala Cys 35 40 45 Ser Phe Asp Leu Cys AsnSer Tyr Asp Val Leu Ser Ala Leu Ser Gly 50 55 60 Asn Leu Lys Leu Cys PheCys Gly Asp Tyr Ile Phe Ser Glu Glu Ala 65 70 75 80 Gln Val Lys Asp ValPro Val Val Thr Ser Val Thr Thr Ala Gly Val 85 90 95 Gly Pro Ser Pro AspIle Thr Ser Thr Thr Lys Thr Arg Asn Phe Asp 100 105 110 Leu Val Asn CysAsn Leu Asn Thr Asn Cys Val Ala Val Ala Phe Ser 115 120 125 Leu Pro AspArg Ser Leu Ser Ala Ile Pro Leu Phe Asp Val Ser Phe 130 135 140 Glu ValLys Val Gly Gly Leu Lys Gln Tyr Tyr Arg Leu Pro Met Asn 145 150 155 160Ala Tyr Arg Asp Phe Thr Ser Glu Pro Leu Asn Ser Glu Ser Glu Val 165 170175 Thr Asp Gly Met Ile Glu Val Gln Ser Asn Tyr Gly Phe Val Trp Asp 180185 190 Val Ser Leu Lys Lys Val Ile Trp Lys Asp Gly Val Ser Phe Val Gly195 200 205 Val Gly Ala Asp Tyr Arg His Ala Ser Cys Pro Ile Asp Tyr IleIle 210 215 220 Ala Asn Ser Gln Ala Asn Pro Glu Val Phe Ile Ala Asp SerAsp Gly 225 230 235 240 Lys Leu Asn Phe Lys Glu Trp Ser Val Cys Val GlyLeu Thr Thr Tyr 245 250 255 Val Asn Asp Tyr Val Leu Pro Tyr Leu Ala PheSer Ile Gly Ser Val 260 265 270 Ser Arg Gln Ala Pro Asp Asp Ser Phe LysLys Leu Glu Asp Arg Phe 275 280 285 Thr Asn Leu Lys Phe Lys Val Arg LysIle Thr Ser Ser His Arg Gly 290 295 300 Asn Ile Cys Ile Gly Ala Thr AsnTyr Val Ala Asp Asn Phe Phe Tyr 305 310 315 320 Asn Val Glu Gly Arg TrpGly Ser Gln Arg Ala Val Asn Val Ser Gly 325 330 335 Gly Phe Gln Phe 3403 393 PRT C. trachomatis 3 Met Lys Lys Leu Leu Lys Ser Val Leu Val PheAla Ala Leu Ser Ser 1 5 10 15 Ala Ser Ser Leu Gln Ala Leu Pro Val GlyAsn Pro Ala Glu Pro Ser 20 25 30 Leu Met Ile Asp Gly Ile Leu Trp Glu GlyPhe Gly Gly Asp Pro Cys 35 40 45 Asp Pro Cys Ala Thr Trp Cys Asp Ala IleSer Met Arg Val Gly Tyr 50 55 60 Tyr Gly Asp Phe Val Phe Asp Arg Val LeuLys Thr Asp Val Asn Lys 65 70 75 80 Glu Phe Gln Met Gly Ala Lys Pro ThrThr Asp Thr Gly Asn Ser Ala 85 90 95 Ala Pro Ser Thr Leu Thr Ala Arg GluAsn Pro Ala Tyr Gly Arg His 100 105 110 Met Gln Asp Ala Glu Met Phe ThrAsn Ala Ala Cys Met Ala Leu Asn 115 120 125 Ile Trp Asp Arg Phe Asp ValPhe Cys Thr Leu Gly Ala Thr Ser Gly 130 135 140 Tyr Leu Lys Gly Asn SerAla Ser Phe Asn Leu Val Gly Leu Phe Gly 145 150 155 160 Asp Asn Glu AsnGln Lys Thr Val Lys Ala Glu Ser Val Pro Asn Met 165 170 175 Ser Phe AspGln Ser Val Val Glu Leu Tyr Thr Asp Thr Thr Phe Ala 180 185 190 Trp SerVal Gly Ala Arg Ala Ala Leu Trp Glu Cys Gly Cys Ala Thr 195 200 205 LeuGly Ala Ser Phe Gln Tyr Ala Gln Ser Lys Pro Lys Val Glu Glu 210 215 220Leu Asn Val Leu Cys Asn Ala Ala Glu Phe Thr Ile Asn Lys Pro Lys 225 230235 240 Gly Tyr Val Gly Lys Glu Phe Pro Leu Asp Leu Thr Ala Gly Thr Asp245 250 255 Ala Ala Thr Gly Thr Lys Asp Ala Ser Ile Asp Tyr His Glu TrpGln 260 265 270 Ala Ser Leu Ala Leu Ser Tyr Arg Leu Asn Met Phe Thr ProTyr Ile 275 280 285 Gly Val Lys Trp Ser Arg Ala Ser Phe Asp Ala Asp ThrIle Arg Ile 290 295 300 Ala Gln Pro Lys Ser Ala Thr Ala Ile Phe Asp ThrThr Thr Leu Asn 305 310 315 320 Pro Thr Ile Ala Gly Ala Gly Asp Val LysThr Gly Ala Glu Gly Gln 325 330 335 Leu Gly Asp Thr Met Gln Ile Val SerLeu Gln Leu Asn Lys Met Lys 340 345 350 Ser Arg Lys Ser Cys Gly Ile AlaVal Gly Thr Thr Ile Val Asp Ala 355 360 365 Asp Lys Tyr Ala Val Thr ValGlu Thr Arg Leu Ile Asp Glu Arg Ala 370 375 380 Ala His Val Asn Ala GlnPhe Arg Phe 385 390 4 345 PRT C. pneumonia 4 Met Asn Ser Lys Met Leu LysHis Leu Arg Leu Ala Thr Leu Ser Phe 1 5 10 15 Ser Met Phe Phe Gly IleVal Ser Ser Pro Ala Val Tyr Ala Leu Gly 20 25 30 Ala Gly Asn Pro Ala AlaPro Val Leu Pro Gly Val Asn Pro Glu Gln 35 40 45 Thr Gly Trp Cys Ala PheGln Leu Cys Asn Ser Tyr Asp Leu Phe Ala 50 55 60 Ala Leu Ala Gly Ser LeuLys Phe Gly Phe Tyr Gly Asp Tyr Val Phe 65 70 75 80 Ser Glu Ser Ala HisIle Thr Asn Val Pro Val Ile Thr Ser Val Thr 85 90 95 Thr Ser Gly Thr GlyThr Thr Pro Thr Ile Thr Ser Thr Thr Lys Asn 100 105 110 Val Asp Phe AspLeu Asn Asn Ser Ser Ile Ser Ser Ser Cys Val Phe 115 120 125 Ala Thr IleAla Leu Gln Glu Thr Ser Pro Ala Ala Ile Pro Leu Leu 130 135 140 Asp IleAla Phe Thr Ala Arg Val Gly Gly Leu Lys Gln Tyr Tyr Arg 145 150 155 160Leu Leu Pro Leu Asn Ala Tyr Arg Asp Phe Thr Ser Asn Pro Leu Asn 165 170175 Ala Glu Ser Glu Val Thr Asp Gly Leu Ile Glu Val Gln Ser Asp Tyr 180185 190 Gly Ile Val Trp Gly Leu Ser Leu Gln Lys Val Leu Trp Lys Asp Gly195 200 205 Val Ser Phe Val Gly Val Ser Ala Asp Tyr Arg His Gly Ser SerPro 210 215 220 Ile Asn Tyr Ile Ile Val Tyr Val Lys Ala Asn Pro Glu IleTyr Phe 225 230 235 240 Asp Ala Thr Asp Gly Asn Leu Ser Tyr Lys Glu TrpSer Ala Ser Ile 245 250 255 Gly Ile Ser Thr Tyr Leu Asn Asp Tyr Val LeuPro Tyr Ala Ser Val 260 265 270 Ser Ile Gly Asn Thr Ser Arg Lys Ala ProSer Asp Ser Phe Thr Glu 275 280 285 Leu Glu Lys Trp Phe Thr Asn Phe LysPhe Lys Ile Arg Lys Ile Thr 290 295 300 Asn Phe Asp Arg Val Asn Phe CysPhe Gly Thr Thr Cys Cys Ile Ser 305 310 315 320 Asn Asn Phe Tyr Tyr SerVal Glu Gly Arg Trp Gly Tyr Gln Arg Ala 325 330 335 Ile Asn Ile Thr SerGly Leu Gln Phe 340 345 5 340 PRT C. trachomatis (L2) 5 Met Ser Ser LysLeu Val Asn Tyr Leu Arg Leu Thr Phe Leu Ser Phe 1 5 10 15 Leu Gly IleAla Ser Thr Ser Leu Asp Ala Met Pro Ala Gly Asn Pro 20 25 30 Ala Phe ProVal Ile Pro Gly Ile Asn Ile Glu Gln Lys Asn Ala Cys 35 40 45 Ser Phe AspLeu Cys Asn Ser Tyr Asp Val Leu Ser Ala Leu Ser Gly 50 55 60 Asn Leu LysLeu Cys Phe Phe Gly Asp Tyr Ile Phe Ser Glu Glu Ala 65 70 75 80 Gln ValLys Asp Val Pro Val Val Thr Ser Val Thr Thr Cys Gly Ile 85 90 95 Gly ProSer Pro Asn Ile Thr Ser Thr Thr Lys Thr Arg Asn Phe Asp 100 105 110 LeuVal Asn Cys Asn Leu Asn Glx Asn Cys Ala Ala Val Ala Phe Ser 115 120 125Leu Pro Asp Arg Ser Leu Ser Ala Ile Pro Leu Phe Asp Val Ser Phe 130 135140 Glu Val Lys Val Gly Gly Leu Lys Gln Tyr Tyr Arg Leu Pro Met Asn 145150 155 160 Ala Tyr Arg Asp Phe Thr Ser Glu Pro Leu Asn Ser Glu Ser GluVal 165 170 175 Thr Asp Gly Met Ile Glu Val Gln Ser Asn Tyr Gly Phe ValTrp Asp 180 185 190 Val Ser Leu Lys Lys Val Ile Trp Lys Asp Gly Val SerPhe Val Gly 195 200 205 Val Gly Ala Asp Tyr Arg His Ala Ser Cys Pro IleAsp Tyr Ile Ile 210 215 220 Ala Asn Ser Gln Ala Asn Pro Glu Val Phe IleAla Asp Ser Asp Gly 225 230 235 240 Lys Leu Asn Phe Lys Glu Trp Ser ValCys Val Gly Leu Thr Thr Tyr 245 250 255 Val Asn Asp Tyr Val Leu Pro TyrLeu Ala Phe Ser Ile Gly Ser Val 260 265 270 Ser Arg Gln Ala Pro Asp AspSer Phe Lys Lys Leu Glu Asp Arg Phe 275 280 285 Thr Asn Leu Lys Phe LysVal Arg Lys Ile Thr Ser Ser His Arg Gly 290 295 300 Asn Ile Cys Ile GlyAla Thr Asn Tyr Ile Ala Asp Asn Phe Phe Tyr 305 310 315 320 Asn Val GluGly Arg Trp Gly Ser Gln Arg Ala Val Asn Val Ser Gly 325 330 335 Gly PheGln Phe 340 6 340 PRT C. trachomatis (C) 6 Met Ser Ser Lys Leu Val AsnTyr Leu Arg Leu Thr Phe Leu Ser Phe 1 5 10 15 Leu Gly Ile Ala Ser ThrSer Leu Asp Ala Met Pro Ala Gly Asn Pro 20 25 30 Ala Phe Pro Val Ile ProGly Ile Asn Ile Glu Gln Lys Asn Ala Cys 35 40 45 Ser Phe Asp Leu Cys AsnSer Tyr Asp Val Leu Ser Ala Leu Ser Gly 50 55 60 Asn Leu Lys Leu Cys PheCys Gly Asp Tyr Ile Phe Ser Glu Glu Ala 65 70 75 80 Gln Val Lys Asp ValPro Val Val Thr Ser Met Thr Thr Ala Gly Val 85 90 95 Gly Pro Ser Pro AspIle Thr Ser Thr Thr Lys Thr Arg Asn Phe Asp 100 105 110 Leu Val Asn CysAsn Leu Asn Thr Asn Cys Val Ala Val Ala Phe Ser 115 120 125 Leu Pro AspArg Ser Leu Ser Ala Ile Pro Leu Phe Asp Val Ser Phe 130 135 140 Glu ValLys Val Gly Gly Leu Lys Gln Tyr Tyr Arg Leu Pro Met Asn 145 150 155 160Ala Tyr Arg Asp Phe Thr Ser Glu Pro Leu Asn Ser Glu Ser Glu Val 165 170175 Thr Asp Gly Met Ile Glu Val Gln Ser Asn Tyr Gly Phe Val Trp Asp 180185 190 Val Ser Leu Lys Lys Val Ile Trp Lys Asp Gly Val Ser Phe Val Gly195 200 205 Val Gly Ala Asp Tyr Arg His Ala Ser Cys Pro Ile Asp Tyr IleIle 210 215 220 Ala Asn Ser Gln Ala Asn Pro Glu Val Phe Ile Ala Asp SerAsp Gly 225 230 235 240 Lys Leu Asn Phe Lys Glu Trp Ser Val Cys Val GlyLeu Thr Thr Tyr 245 250 255 Val Asn Asp Tyr Val Leu Pro Tyr Leu Ala PheSer Ile Gly Ser Val 260 265 270 Ser Arg Gln Ala Pro Asp Asp Ser Phe LysLys Leu Glu Asp Arg Phe 275 280 285 Thr Asn Leu Lys Phe Lys Val Arg LysIle Thr Ser Ser His Arg Gly 290 295 300 Asn Ile Cys Ile Gly Ala Thr AsnTyr Ile Ala Asp Asn Phe Phe Tyr 305 310 315 320 Asn Val Glu Gly Arg TrpGly Ser Gln Arg Ala Val Asn Val Ser Gly 325 330 335 Gly Phe Gln Phe 340

That which is claimed is:
 1. A method for inducing immunity againstChlamydia in a mammalian subject, the method comprising: administeringto a mammalian subject a PorB vaccine in an amount sufficient to elicitan immune response; wherein said immune response is sufficient todecrease risk of onset of disease caused by Chlamydia.
 2. The method ofclaim 1, wherein the vaccine comprises a PorB polypeptide of Chlamydiatrachomatis.
 3. The method of claim 1, wherein the vaccine comprises aPorB polypeptide of Chlamydia pneumoniae.
 4. The method of claim 1,wherein the vaccine comprises a PorB polypeptide of Chlamydia psittaci.5. The method of claim 1, wherein the vaccine comprises nucleic acidencoding a PorB polypeptide for expression in the subject.
 6. The methodof claim 1, wherein the vaccine comprises a recombinant live vaccinewhich comprises a PorB polypeptide-encoding polynucleotide.
 7. Themethod of claim 6, wherein the live vaccine is a recombinant viruscomprising a PorB polypeptide-encoding polynucleotide for expression inthe subject.
 8. The method of claim 1, wherein the vaccine comprises aPorB polypeptide and said administering is subcutaneous, intramuscular,intradermal, or intravenous.
 9. The method of claim 1, wherein thevaccine comprises a nucleic acid encoding a PorB polypeptide and whereinsaid administering subcutaneous, transdermal, subdermal, intradermal,topical, or intramuscular.
 10. A vaccine composition comprising: anisolated PorB polypeptide in an amount effective to induce an immuneresponse in a subject; and a pharmaceutically acceptable carrier. 11.The vaccine composition of claim 1 10, wherein the PorB polypeptide is aPorB polypeptide of C. trachomatis.
 12. The vaccine composition of claim10, wherein the PorB polypeptide is aPorB polypeptide of C. pneumoniae.13. The vaccine composition of claim 10, wherein the PorB polypeptide isa PorB polypeptide of C. psittaci.
 14. A vaccine composition comprising:an isolated polynucleotide comprising a sequence encoding a PorBpolypeptide, the polynucleotide being present in an amount effective toprovide forproduction of the PorB polypeptide in a host in an amountsufficient to induce an immune response in a subject; and apharmaceutically acceptable carrier.
 15. The vaccine composition ofclaim 14, wherein the PorB polypeptide is a PorB polypeptide of C.trachomatis.
 16. The vaccine composition of claim 14, wherein the PorBpolypeptide is a PorB polypeptide of C. pneumoniae.
 17. The vaccinecomposition of claim 14, wherein the PorB polypeptide is a PorBpolypeptide of C. psittaci.
 18. A method of providing a subject withprotective immunity to chlamydial infection, the method comprising:administering to a subject an amount of an antibody that specificallybinds a PorB polypeptide, the amount being effective to inhibit ormitigate chlamydial infection in the subject.
 19. A method fordetermining exposure of a subject to a Chlamydia infection, the methodcomprising: contacting a biological sample from a subject suspectedhaving been exposed to Chlamydia or having a Chlamydia infection with a)an antibody that specifically binds a PorB polypeptide or b) a PorBpolypeptide, said contacting being under conditions sufficient to allowfor formation ofspecific antibody-PorB polypeptide binding pairs; anddetecting the antibody-PorB binding pairs; wherein detection of theantibody-PorB binding pairs indicates the subject has been exposed toinfection by Chlamydia.
 20. A method for detecting a Chlamydia infectionin a subject, the method comprising: contacting a test sample from asubject suspected of having a chlamydial infection with a probe, theprobe being selected from the group consisting of a) a PorB polypeptidethat can be bound by a PorB-specific antibody, b) a specific PorBantibody; and c) a PorB polypeptide-encoding polynucleotide; anddetecting binding of the probe to the test sample; wherein detection ofbinding of the probe to the test sample is indicative of Chlamydiainfection in the subject.
 21. The method of claim 20, wherein the probeis an anti-PorB antibody for detection of a PorB polypeptide in the testsample.
 22. The method of claim 20, wherein the probe is a PorBpolypeptide for detection of an anti-PorB antibody in the test sample.23. The method of claim 20, wherein the probe is a PorB polynucleotidefor detection of a PorB polypeptide-encoding polynucleotide in the testsample.
 24. A method for identifying an agent that inhibits PorBfunction, the method comprising: contacting a PorB polypeptide with acandidate agent; and determining the effect of the candidate agent onPorB activity in transport of α-ketoglutarate.
 25. The method of claim24, wherein the PorB polypeptide is present in a membrane.
 26. Themethod of claim 24, wherein the membrane is an artificial membrane. 27.The method of claim 26, wherein the artificial membrane is a membrane ofa liposome.
 28. The method of claim 25, wherein the membrane is anextracellular membrane of a host cell.
 29. A method for identifying anagent that inhibits PorB function, the method comprising: combining aliposome comprising a PorB polypeptide with a candidate agent andα-ketoglutarate; and determining the effect of the candidate agent onPorB activity in transport of α-ketoglutarate; wherein a decrease intransport of α-ketoglutarate indicates that the agent inhibits PorBfunction.